Dietary polyglutamyl folates undergo digestion at the brush border of the intestinal membrane prior to absorption of folic acid derivatives. We characterized the molecular properties of human intestinal folate hydrolase, which is an expression of Glutamate Carboxypeptidase II (GCPII). We identified a H475Y polymorphism in the catalytic site of GCPII, which reduces enzyme activity in cell transfectants and was associated with relatively low folate and elevated homocysteine levels in an aging Caucasian population. Also we found an exon 18-deletion splice variant (GCP-18) that is expressed together with wild type GCPII in normal human jejunal mucosa and universally in duodenal biopsies from patients with various gastrointestinal disorders. We propose that the H475Y allele reduces folate absorption resulting in lower folate levels and hyperhomocysteinemia, while both H475Y and GCP-18 variants modulate the expression of GCPII in the intestine. The overall goal of the proposed studies is to determine the potential significance of the H475Y allele in clinical conditions associated with hyperhomocysteinemia and to study the regulation of GCPII transcription and expression in mammalian cell models. Specific Aim I will address the incidence and clinical significance of the H475Y allele in parents and affected children with neural tube defects, in aging subjects with altered cognition including Alzheimer's disease, and in patients with known coronary artery disease. DNA samples from these patient groups and controls will be screened for H475Y and potential homozygotes by a specific PCR-restriction enzyme method and incidence will be correlated with disease diagnosis, folate and homocysteine levels. Additional polymorphisms will be sought in non- Caucasian subjects within these groups. Specific Aim II will utilize a human intestinal Caco-2 cell line known to express GCPII in order to study transcription regulation using promoter regions of different length and luciferase reporter. Additional studies of post-transcriptional regulation will utilize both a separate Cos-7 mammalian cell line known not to express GCPII and intestinal Caco-2 cells, and cell transfection and co-transfection with GCPII, H475Y, or GCP-18 variants. Cellular trafficking and membrane insertions will be studies after labeling each form with green and red fluorescent tags, and protein interactions will be studied by immunoprecipitation using separate antibodies to each tag. Overall, the proposed studies will enhance understanding of the central role of GCPII in regulating folate availability and the clinical significance of a novel polymorphism in GCPII associated with hyperhomocysteinemia.